Many neurological and all neurodegenerative diseases are caused by death of neurons or loss of their neuritis. Currently, there are no drugs that are neuroprotective or neurorestorative. Several proteins supporting neuronal survival have been shown to be effective against neurological and neurodegenerative diseases in animal models and in clinical trials, e.g., GDNF family of ligands (GLFs) in Parkinson's disease and chronic pain. However, proteins are large molecules with poor pharmacokinetic properties and are cannot penetrate the blood-brain barrier.
Neurons, as non-dividing cells, require constant survival signals from neighboring cells, from the extracellular matrix (ECM) and from the environment, to remain alive. The “stay alive” signals are usually carried by neurotrophic factors promoting neuronal survival. In certain pathological conditions, such as Parkinson's (PD) and Alzheimer's diseases, neurons progressively degenerate. Neurons loose synaptic contacts, undergo axonal degeneration, and eventually die.
Currently available therapies for neurodegenerative diseases are symptomatic, and there are no other available treatments that can reverse or significantly slow down the neurodegeneration. The neurotrophic factor-based therapies hold a great promise, because in addition to the promotion of neuronal survival they also induce axonal regeneration, support the formation of synapses and stimulate functional properties of neurons.
Glial cell line-derived neurotrophic factor (GDNF) is a distant member of the transforming growth factor β superfamily and a founding member of the GDNF family ligands (GFL). This family consists of four members: GDNF, neurturin (NRTN), artemin (ARTN) and persephin (PSPN) (FIG. 1), all of which are potent neurotrophic factors (Airaksinen and Saarma, 2002). Since its discovery in 1993, GDNF has attracted substantial attention for its ability to support the survival of dopaminergic neurons, induce axonal sprouting and regulate functional dopamine metabolism in these neurons, which degenerate in PD (Lin et al., 1993). In addition, GDNF is one of the few growth factors that not only protects, but also repairs dopamine neurons in animal models of PD (Bespalov and Saarma, 2007; Lindholm et al., 2007).
Although GDNF has already shown protective and neurorestorative effects in a number of animal models of Parkinson's disease and demonstrated very promising results in two clinical trials (Gill et al., 2003; Slevin et al., 2005), a recent study failed to show clear clinical benefits of GDNF (Lang et al., 2006). This discrepancy might be explained by differences in trial setups, disease state of the patients and the properties of E. coli expressed GDNF. To date, there is no clear understanding why these three trials resulted in different outcomes.
GDNF may also be important for the treatment of amyotrophic lateral sclerosis (ALS) as GDNF is supportive for motoneurons (Henderson et al., 1994). For the treatment of depression as RET signaling increases amount of dopamine in the brain (Mijatovic et al., 2007). GDNF or its mimetics could also be used as male contraceptives (Meng et al., 2000).
NRTN is a very promising molecule as recent Phase II clinical trials with intraputamenal injections of adenovirus bearing NRTN gene demonstrated a significant improvement in Parkinson's disease patients (Ceregene Inc., press release). ARTN is being tested for neuropathy in Phase I trials conducted by Biogen Idec/NsGene as it was demonstrated to be efficient in animal model of chronic pain (Gardell et al., 2003) and being restorative for sensory neurons (Wang et al., 2008). PSPN is considered for the treatment of stroke and Alzheimer's disease (Golden et al., 2003; Tomac et al., 2002)
While the GFLs-receptor complex is considered as an adequate drug target, the GFLs polypeptides are probably the inappropriate pharmacological agents. One hurdle in protein-based therapies is bioavailability. GDNF is a basic protein of 134 amino acids, which is unable to penetrate the blood-brain barrier. Therefore, brain surgery is required to deliver it. Furthermore, GDNF, NRTN and ARTN interact with heparan sulfates—the components of the extracellular matrix (ECM) (Lin et al., 1993). This interaction dramatically reduces the diffusion of GFLs from the area of its application or production. Recombinant GDNF may induce inflammation and formation of anti-GDNF antibodies (Lang et al., 2006) and the price of recombinant GDNF is high. The properties of E. coli-produced recombinant GDNF may vary from batch to batch, since it is first produced as an inactive protein followed by in vitro renaturation. Finally, GDNF is promiscuous; not only can it activate RET through GFRα1 (weakly also through GFRα2 and GFRα3), but GDNF can also activate completely different receptors: neural cell adhesion molecule NCAM and syndecan glycoproteins that carry GDNF-binding heparan sulphate side chains (Bespalov et al., unpublished) (Sariola and Saarma, 2003). These pleiotropic GDNF actions can lead to multiple side-effects.
Since mammalian cells secrete active GDNF under strict quality control, gene- and cell therapy approach may help to overcome the immunogenic and inflammation response problems associated with E. coli-produced recombinant GDNF. Viral vectors and implantable devices containing polymer-encapsulated genetically modified cells that secrete GDNF (Sautter et al., 1998) could be used in the therapy of Parkinson's disease. Unfortunately, these strategies may increase the risk of cancer, because constant RET activation by unregulated and continuously produced GDNF may lead to malignancy. For instance, GDNF-overexpressing transgenic mice develop testicular cancer (Meng et al., 2001). Unlike gene- or cell therapy, small molecules do not trigger permanent RET activation, since they are delivered at defined time intervals and they are rapidly degraded in the organism. The risk of carcinogenesis is further reduced by the fact that GDNF-mimetics are partial agonists.